A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae

Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10% (exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases exhibited identical mobility, each one consisting of two polypeptides with M _r of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts. These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond the permeability barrier of the cell.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 2. Effect of VIP on cAMP production and on cell-surface VIP-binding sites

Vasoactive intestinal peptide (VIP) stimulated in a dose-dependent manner the accumulation of cAMP in human melanoma-derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half-maximum cAMP production was obtained at 0.78 nM VIP. VIP-related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP much greater than peptide histidine-methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin greater than glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production. After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 less than 2 min) and homologous. Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP-binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37 degrees C with a VIP concentration of 1 microM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half-maximum decrease in binding of mono[125I]iodinated VIP (125I-VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP-binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP-free medium. The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.     

Localization of the nerve growth factor receptor on fetal human schwann cells in culture

Previous studies have established that Schwann cells (SC) in culture express an NGF receptor. In this study, cultures of fetal human SC were established from fetal nerves and various light microscopic (LM) and electron microscopic (EM) techniques were used to localize the NGF receptor on the SC. Results indicate that NGF receptor is localized to the plasma membrane of the SC. Quantitative digital analysis determined that the distal portion of the SC process had high concentrations of NGF receptor. The possible functional significance of this latter observation is discussed in terms of SC migration and ensheathment of axons.     

Rapid postnatal changes in F1-ATPase proteins and in the uncoupling protein in brown adipose tissue mitochondria of the newborn rat

Changes in F1-ATPase and UCP protein contents and in the activity of respiratory complexes I, II and IV of brown adipose tissue mitochondria are reported during the first 0-6 hours of life in the rat. Mitochondrial UCP/F1-ATPase protein ratio is used to define the onset of thermogenic differentiation of brown adipose tissue mitochondria. It is concluded that mitochondrial differentiation occurs soon after birth and that the process is accelerated by hypothermic conditions.     

Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures

Dissociation of protein-containing structures by modification of protein amino groups with dicarboxylic acid anhydrides is a mild procedure which, in some cases, offers advantages over treatment with alternative dissociating agents, such as urea, guanidine hydrochloride, detergents, high ionic strength, and extremes of pH: In addition to dissociating multimeric proteins and protein aggregates, dicarboxylic acid anhydrides are effective dissociating agents for membrane-bound proteins and nucleoprotein particles. With most dicarboxylic acid anhydrides reviewed, the introduced reagent residues can be eliminated under moderate acid conditions, which allows the purification of unmodified individual components, and the use of disassembly-reconstitution systems valuable for investigating the structural and functional roles played by the individual components of complex particles:Each reagent can be suitable for a particular purpose, depending on the required specificity of the modification and stability of the modified groups: The stability of the acylated amino groups ranges from the very stable succinylated amino groups to the very labile acylation obtained with dimethylmaleic anhydride: Between these extremes, the stability of the modified amino groups decreases stepwise in the following order: maleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, citraconic, and 3,4,5,6-tetrahydrophthalic anhydride. With respect to the selectivity of the produced modification, little or no modification of hydroxyamino acid and cysteine residues has been observed with dimethylmaleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, and 3,4,5,6-tetrahydrophthalic anhydrides: With the other reagents, the extent of modification of hydroxyamino acid residues increases in the order citraconic, maleic and succinic anhydride: Citraconic and maleic anhydrides can produce irreversible modification of cysteine residues, the reactivity of sulfhydryl groups being higher with maleic anhydride:     

Influence of the solvent on the conformational-dependent properties of random coil polypeptides. II. Mean square optical anisotropies and Kerr constants

Mean square optical anisotropies and molar Kerr constants were calculated for homopolypeptides of the 20 natural amino acids and of several enzymes and proteins in the random-coil state. The effect of hydration was taken into account in constructing the molecular potential that gives the conformational energies as a function of the rotational angles phi and psi of the backbone and chi(1) of the side chain. The Rotational Isomeric State model was used in calculated energies, the Valence Optical Scheme and the matrix calculus technique of Flory being employed in the evaluation of the optical properties. The results are compared with calculations for the same substances that were performed without taking into account the solvent, as well as with other similar studies. The Kerr constant is confirmed as being one of the most sensitive properties of a given polypeptide to the residue class and to the sequence of those residues.     

Ca2+ ionophores inhibit superoxide generation by chemotactic peptide in rabbit neutrophils and the correlation with intracellular calcium

Summary Effects of Ca²⁺ ionophores, A23187 and lasalocid, on superoxide anion generation by chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine methyl ester, in rabbit peritoneal exudate neutrophils were studied. The ionophores by themselves did not activate superoxide anion generation in these neutrophils. When preincubated with the cells for 2 min, both the ionophores inhibited superoxide generation induced by chemotactic peptide. The inhibition was present even in the absence of extracellular Ca²⁺ and the inhibition was better then. Lasalocid produces a dose-dependent chlortetracycline fluorescence decrease response in neutrophils loaded with chlortetracycline. This response is independent of extracellular Ca²⁺ concentration and is related to release of Ca²⁺ from intracellular storage sites. The dose-range at which lasalocid gives this response is same as the dose-range at which it causes inhibition of superoxide response. It may be concluded that the inhibition of superoxide generation by these ionophores is correlated to intracellular Ca²⁺ modulation.Abbreviations FMLP Formyl-Methionyl-Leucyl-Phenylalanine methyl ester     

Metabolic adaptation of the renal carbohydrate metabolism. I. Effects of starvation on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules

Summary The influence of starvation on renal carbohydrate metabolism was studied in the proximal and distal fragments of the nephron. Starvation induced a double and opposite adaptation mechanism in both fractions of the renal tubule. In renal proximal tubules, the gluconeogenic flux was stimulated progressively during a period of 48 hours of starvation (2.15 fold), due, in part, to a significant increase in the fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase activities although with different characteristics. Fructose 1,6-bisphosphatase activity from this tubular fragment increased only at subsaturating subtrate concentration (68%) which involved a significant decrease in the Km (35%) for fructose 1,6-bisphosphate while there was no change in Vmax. This behaviour clearly indicates that it is related to modifications in the activity of the preexistent enzyme in the cell. Proximal phosphoenolpyruvate carboxykinase activity increased proportionally at both substrate concentrations (86 and 89% respectively) which brought about changes in Vmax without changes in Kin, all of which are in accordance with variations in the cellular levels of the enzyme. In the renal distal tubules, the glycolytic capacity drastically decreased throughout the starvation time. At 48 hours 65% of inhibition was shown. We have found a short term regulation of phosphofructokinase activity by starvation which involves an increase in Km (2.2 fold) without changes in Vmax, as a result of these kinetic changes, an inactivation of phosphofructokinase was detected at subsaturating concentration of fructose 6-phosphate. On the contrary, this nutritional state did not modify the kinetic behaviour of renal pyruvate kinase. Finally, neither proximal glycolytic nor distal gluconeogenic capacities and related enzymes activities were changed during starvation.     

Processing of SP1 precursor in a cell-free system from poly(A+) mRNA of human placenta

Cell-free translation of polyadenylated mRNA from human term placenta in a wheat germ extract, after immunoprecipitation with antibodies directed against purified pregnant serum SP₁, yielded a single polypeptide of 31 kDa. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of two polypeptides, one of them of 46 kDa and the other of 28 kDa. Both polypeptides were protected from limited proteolysis and when the assay was performed with lytic detergent concentrations in addition to proteases, this protection was abolished indicating that the polypeptides were segregated into the microsomal vesicles. The cleavage of a signal peptide of 3 kDa from the 31 kDa primary translation product gives rise to 28 kDa and accounts for the slight increase in electrophoretic mobility. The treatment of the immunoprecipitated products with Endoglycosidase H and -mannosidase, suggested that only the 46 kDa polypeptide is a glycoprotein.From the results obtained we conclude that SP₁ is synthesized and processed to a glycoprotein of 46 kDa which would be a protomeric form of the oligomers reported in pregnant serum by other authors.Abbreviations PMSF phenylmethyl sulfonylfluoride - TCA trichloroacetic acid - DTT dithiotreitol     

Degradation of diarylethane structures by Pseudomonas fluorescens biovar I

Pseudomonas fluorescens biovar I was isolated from a pulp mill effluent based on its ability to grow on synthetic media containing 1,2-diarylethane structures as the sole carbon and energy source. Analysis of samples taken from cultures of this strain in benzoin or 4,4-dimethoxybenzoin (anisoin), showed that cleavage between the two aliphatic carbons takes place prior to ring fission. Intermonomeric cleavage was also obtained with crude extracts. Substrates of this reaction were only those 1,2-diarylethane compounds that supported growth of the bacterium. The purification and partial characterization of an enzyme that catalyzes the NADH-dependent reduction of the carbonyl group of benzoin and anisoin is also reported.